Virginia E. Davis

Dopamine-derived tetrahydroisoquinoline alkaloids—Inhibitors of neuroamine metabolism☆

Abstract The formation of salsolinol and tetrahydropapaveroline (THP) in vitro as aberrant metabolites of dopamine has been established previously. Demonstrating the formation of these compounds in vivo is complicated by the probability that they are extensively metabolized. The present studies indicate that a primary metabolic route for these compounds may be methylation of one or more of the free hydroxyl groups catalyzed by catechol-O-methyl transferase (COMT). Through the use of a partially purified COMT preparation from rat liver, the maximal velocities of salsolinol and THP O-methylation proved to be three to five times the maximal velocities of norepinephrine and dopamine O-methylation. The Michaelis constants determined for salsolinol, norepinephrine and dopamine are similar, whereas the Km of COMT for THP (0.03 mM) is approximately one-tenth that of the above substrates. Salsolinol and THP are competitive inhibitors of dopamine O-methylation in vitro, their calculated inhibitor constants (ki values) being 0.13 and 0.02 mM respectively. The effects of these two alkaloids on rat brain monoamine oxidase (MAO) activity were also measured. They were found to be equally potent substrate competitive inhibitors of rat brain MAO in vitro, with calculated Ki values of 0.14 mM (salsolinol) and 0.20 mM (THP). These data suggest that, if formed in vivo under certain pharmacological conditions, aberrant neuroamme derived alkaloids may alter the metabolic disposition of endogenous neuroamines with resultant modification of adrenergic function.

A rapid and sensitive potentiometric assay for monoamine oxidase using an ammonia-selective electrode.

A sensitive and convenient method for the estimation of monoamine oxidase (MAO) activity using an ammonia-selective electrode has been developed. The ammonia in the sample diffuses through the gas-permeable membrane until NH3-N partial pressures are at equilibrium, and the resulting potential (in millivolts) is recorded with a millivolt/pH meter. The amount of ammonia generated enzymatically is obtained by calculating the difference in ammonia concentration in control and test incubation mixtures at pH 12.0. The sensitivity of this method is comparable to radiometric, fluorometric, and polarographic techniques. The millivolt potential corresponding to ammonia formed from substrate by MAO in brain homogenate and mitochondrial preparations is linear to enzyme protein concentration and to time during the 30-min incubation period. The stoichiometric relationship between the amounts of ammonia production and amine depletion is evidenced by the equal amounts of ammonia formed and substrate depleted. The assay technique offers diverse utility. Numerous primary amine substrates can be employed with this method. The procedure is also useful for the study of enzyme-inhibitor interactions. Compared to many other assay procedures, the method outlined offers the advantages of precision, sensitivity, accuracy, and rapidity.

Inhibition of catecholamine uptake and retention in synaptosomal preparations by tetrahydroisoquinoline and tetrahydroprotoberberine alkaloids

Abstract The effects of racemic mixtures of representative tetrahydroisoquinoline and tetrahydroprotoberberine alkaloids on the mechanisms of catecholamine uptake and retention were studied in synaptosomal preparations from whole rat brain. The synaptosomes were incubated with ( 14 C )-d,l- norepinephrine or ( 14 C)-dopamine in the presence of various concentrations of salsolinol (SAL), tetrahydropapaveroline (THP), 2,3,10,11-tetrahydroxyberbine (THB), or 2,3,10,11-tetramethoxyberbine (TMB). Levels of radioactivity in synaptosomes preloaded with labeled norepinephrine were significantly diminished by the addition of 10 −4 M THP, THB or SAL to approximately 42.7 per cent (P −5 M, also significantly decreased synaptosomal retention of the labeled neuroamine. ( 14 C)-dopamine was used in an analysis of alkaloid effects on catecholamine uptake kinetics. K i values obtained were: 0·7 × 10 −5 M (THP); 3·5 × 10 −5 M (THB); 1·25 × 10 −4 M (SAL); and 2·2 × 10 −4 M (TMB). These results have been interpreted to suggest that the affinity of these amine-derived alkaloids for the catecholaminergic uptake mechanisms, although not marked when compared to that of dopamine or norepinephrine, may be sufficient under conditions of highly localized formation and accumulation to have important physiological sequelae.

Neuroamine-derived alkaloids: Substrate-preferred inhibitors of rat brain monoamine oxidase in vitro

The effects of tetrahydropapaveroline (THP), salsolinol (SAL) and various hydroxylated and methoxylated tetrahydroprotoberberine (THPB) alkaloids on monoamine oxidase (MAO) forms A and B in rat brain homogenates were investigated. The substrates utilized were serotonin, a specific substrate for type A MAO; tyramine, a substrate for both type A and B MAO; and benzylamine, a preferred substrate for type B MAO. The concentrations of THP, SAL, 2,3,9,10-tetrahydroxyberbine and 2,3,10,11-tetrahydroxyberbine producing 50 per cent inhibition (I50) of the oxidation of serotonin were 1.0 mM, 0.25 mM, 0.24 mM and 0.04 mM, respectively. In marked contrast, the I50 concentrations of these alkaloids with benzylamine as substrate were 4.4 mM, 50 mM, 5.6 mM and 13 mM, respectively. These findings indicated that SAL and the tetrahydroxyberbines were substrate-preferred inhibitors of type A MAO whereas THP was a relatively nonspecific inhibitor of rat brain MAO. Kinetic data revealed that THP, SAL and 2,3,10,11-tetrahydroxyberbine inhibited the oxidation of serotonin in a typical competitive manner with apparent Ki values of 0.82 mM, 0.11 mM and 0.05 mM, respectively. THP and SAL noncompetitively inhibited benzylamine oxidation with apparent Ki values of 5.0 mM and 52 mM, respectively, while 2,3,10,11-tetrahydroxyberbine competitively inhibited the oxidation of benzylamine with an apparent Ki of 3.8 mM. Sequential replacement of the hydroxyl groups at the 2,3,9,10 and 11 positions of the berbine ring system by methoxyl groups substantially decreased the potency and selectivity of MAO inhibition. The interaction of these alkaloids with the metabolic pathways of neurotransmitters suggests that these compounds may be of relevance in the modification of central synaptic function.

Identification of catecholamine-derived alkaloids in mammals by gas chromatography and mass spectrometry

Abstract Tetrahydropapaveroline, the tetrahydroisoquinoline alkaloid derived from dopamine, is convertedin vivo by rats and by rat-liver and brain preparations to tetrahydropprotoberberine alkaloids. The latter alkaloids have also been identified for the first time in the urine of parkinsonian patients receiving l -DOPA therapy. These findings suggest that man, like plants, may have the ability to elaborate several classes of alkaloids with potentially important pharmacological consequences.

Stereoselective enzymatic O-methylation of tetrahydropapaveroline and tetrahydroxyberbine alkaloids

Abstract Modification of neuroamine metabolism leading to the formation of isoquinoline alkaloids has been the target of several investigations. This study describes the differential enzymatic O -methylation pattern of the racemates and optical isomers of tetrahydropapaveroline (THP) and 2,3,10,11-tetrahydroxy-berbine (THB) by a rat liver catechol O -methyltransferase (COMT) preparation. Reaction products were separated and isolated by high-pressure liquid chromatography, and structural identity was confirmed by synthesis and gas chromatography/mass spectrometry. The positions of enzymatic O -methylations were markedly influenced by the particular optical isomeric form of substrate employed. The optical isomers and racemates of THP and THB were mainly mono- O -methylated with trace amounts of di- O -methylation. The mono- O -methylations of THP were contained exclusively in the isoquinoline ring at positions 6 or 7, while mono- O -methylations of THB occurred at the vicinal hydroxyl groups of both rings A and D. This enzymatic process was evaluated further by examining the effect of classical inhibitors of COMT on these O -methylations. The results presented demonstrate that vicinal hydroxyl moieties of THP and THB are mono- O -methylated at either of the sites and that the magnitude of the positional isomer product ratio results from the optical isomeric orientation in which the substrate binds to the enzyme.

Identification of 5-Hydroxytryptophol as a Serotonin Metabolite In Man.∗

Summary 5-Hydroxytryptophol has been isolated, identified, and measured in urine of subjects with carcinoid tumors. In the urine of normal subjects and of patients with carcinoid tumors who ingested 5-HT-C14, 2.3% of the excreted C14 was attributable to 5-HTOH and its conjugates and 82.3% to 5-HIAA. These data show that 5-HTOH is a 5-HT metabolite in man as well as in animals.

Dopamine receptor topography: Characterization of antagonist requirements of striatal dopamine-sensitive adenylate cyclase using protoberberine alkaloids

Abstract Representative protoberberine-related alkaloids, i.e. tetrahydroprotoberberines (THPB), quaternary protoberberine salts (Quat. PB) and quaternary dehydroprotoberberine salts (Dehyd. Quat. PB), have been used to characterize the geometric and stereospecific requirements of antagonists of the dopamine receptor. The optical isomers of 2, 3, 10, 11-THPB were tested for their ability to antagonize dopamine stimulated adenylate cyclase activity. The results indicate that (±)-2. 3, 10, 11-THPB inhibited the ability of 100 μM dopamine to elevate adenylate cyclase in homogenates of the rat caudate nucleus. The ic 50 was observed to be 6 μM. The S-(−)-isomer of 2, 3, 10, 11-THPB was a more potent antagonist of dopaminesensitive adenylate cyclase activity than the R-(+)-isomer. The ic 50 for (−)-THPB was 1μM whereas that for the (+)-isomer was 50 μM. The data also show that the positional isomer, 2, 3, 9, 10-THPB, antagonized dopamine activation of adenylate cyclase with the same degree of potency as 2, 3, 10, 11-THPB. Exhaustive O-methylation of THPB at all four hydroxyl positions, the 2, 3-position of the “a” ring and the 10, 11-position of the “d” ring as in xylopinine or the 2, 3-position of the “a” ring and the 9, 10-position of the “d” ring as in tetrahydropalmatine, rendered these compounds weak antagonists of the dopamine response. Selective O-methylation of the THPB molecule markedly altered the potency of the resultant compounds as antagonists depending on the position of the O-methyl substitution. Overall, these data are consistent with the idea that the orientation of the nitrogen atom in a fixed (cis: gauche) position 2 carbon atoms from a catechol nucleus renders antagonist properties to these compounds which interact with the dopamine receptor.

A novel synthesis of Aporhoeadanes

Abstract A one-pot synthesis of aporhoeadanes from the reaction of 3,4-dihydro-isoquinolines, 2-chloromethylbenzoyl chloride and sodium hydroxide, is described.

Isoquinoline alkaloids. Inhibitory actions on cation-dependent ATP-phosphohydrolases.

Representatives of eleven different classes of isoquinoline alkaloids inhibit Na+, K+-ATPase and Mg2+-ATPase in rat brain microsomal preparations. In most cases the Na+, K+-ATPase is more sensitive than Mg2+-ATPase to inhibition by the alkaloids. The classes of alkaloids can be ranked according to potency of inhibition of Na+, K+-ATPase. Protoberberines are most effective, followed in decreasing order by benzophenanthridines, benzylisoquinolines, aporphines, tetrahydroprotoberberines, pavines, protopines, isoquinolines, tetrahydrobenzylisoquinolines, morphinanes, and tetrahydroisoquinolines. As specific representatives of each of the first four classes of alkaloids, berberine, sanguinarine, papaveroline and 1,2,10,11-tetrahydroxyaporphine, respectively, prove most valuable in kinetic studies because they exhibit the greatest inhibitory action on brain Na+, K+-ATPase. Kinetic analyses plotted in double reciprocal form reveal that berberine and 1,2,10,11-tetrahydroxyaporphine are simple linear competitive inhibitors with respect to ATP, whereas sanguinarine and papaveroline are simple linear noncompetitive inhibitors. These four representative alkaloids exhibit nonlinear competitive inhibition with respect to Na+-activation. Additionally, these alkaloids significantly inhibit rat brain microsomal K+-activatedpNPPase. The results demonstrate that certain members of several classes of isoquinoline alkaloids markedly affect various cation-dependent phosphohydrolases in vitro.